Accumulation, persistence and effects of indospicine residues in camels fed Indigofera plant

Indospicine (L-2-amino-6-amidinohexanoic acid) is a natural hepatotoxin found in all parts of some Indigofera plants such as I. linnaei and I. spicata. Several studies have documented a susceptibility to this hepatotoxin in different species of animals, including cattle, sheep, dogs and rats, which are associated with mild to severe liver disease after prolonged ingestion. However, there is little published data on the effects of this hepatotoxin in camels, even though Indigofera plants are known to be palatable to camels in central Australia. The secondary poisoning of dogs after prolonged dietary exposure to residual indospicine in camel muscle has raised additional food safety concerns. In this study, a feeding experiment was conducted to investigate the in vivo accumulation, excretion, distribution and histopathological effects of dietary indospicine on camels. Six young camels (2 – 4 year old), weighing 270 − 390 kg were fed daily a roughage diet consisting of Rhodes grass hay and lucerne chaff, supplemented with Indigofera and steam flaked barley. Indigofera (I. spicata) was offered at 597 mg DM/kg body weight (bw)/day designed to deliver 337 µg indospicine/kg bw/day, and fed for a period of 32 days. Blood and muscle biopsies were collected over the period of the study. Concentrations of indospicine in the plasma and muscle biopsy samples were quantitated by validated ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS). The highest concentrations in plasma (1.01 mg/L) and muscle (2.63 mg/kg fresh weight (fw)) were found at necropsy (day 33). Other tissues were also collected at necropsy and analysis showed ubiquitous distribution of indospicine, with the highest indospicine accumulation detected in the pancreas (4.86 ± 0.56 mg/kg fw) and liver (3.60 ± 1.34 mg/kg fw); followed by the muscle, heart and kidney. Histopathological examination of liver tissue showed multiple small foci of predominantly mononuclear inflammatory cells. After cessation of Indigofera intake, indospicine present in plasma in the remaining 3 camels had a longer terminal elimination half-life (18.6 days) than muscle (15.9 days), and both demonstrated mono-exponential decreases.

Natural Hendra Virus Infection in Flying-Foxes - Tissue Tropism and Risk Factors

Hendra virus (HeV) is a lethal zoonotic agent that emerged in 1994 in Australia. Pteropid bats (flying-foxes) are the natural reservoir. To date, HeV has spilled over from flying-foxes to horses on 51 known occasions, and from infected horses to close-contact humans on seven occasions. We undertook screening of archived bat tissues for HeV by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Tissues were tested from 310 bats including 295 Pteropodiformes and 15 Vespertilioniformes. HeV was detected in 20 individual flying-foxes (6.4%) from various tissues including spleen, kidney, liver, lung, placenta and blood components. Detection was significantly higher in Pteropus Alecto and Pconspicillatus, identifying species as a risk factor for infection. Further, our findings indicate that HeV has a predilection for the spleen, suggesting this organ plays an important role in HeV infection. The lack of detections in the foetal tissues of HeV-positive females suggests that vertical transmission is not a regular mode of transmission in naturally infected flying-foxes, and that placental and foetal tissues are not a major source of infection for horses. A better understanding of HeV tissue tropism will strengthen management of the risk of spillover from flying-foxes to horses and ultimately humans.

Toxoplasmosis in Indo-Pacific humpbacked dolphins (Sousa chinensis) from Queensland

Objective: To describe the clinical signs, gross pathology, serology, bacteriology, histopathology, electron microscopy and immunohistochemistry findings associated with toxoplasmosis in four Indo-Pacific humpbacked dolphins (Sousa chinensis) that stranded in Queensland in 2000 and 2001. Design: Clinical assessment, gross necropsy, and laboratory examinations. Procedure: Necropsies were performed on four S chinensis to determine cause of death. Laboratory tests including serology, bacteriology, histopathology and transmission electron microscopy were done on the four dolphins. lmmunohistochemistry was done on the brain, heart, liver, lung, spleen and adrenal gland from various dolphins to detect Toxoplasma gondii antigens. Results: Necropsies showed all of four S chinensis that stranded in Queensland in 2000 and 2001 had evidence of predatory shark attack and three were extremely emaciated. Histopathological examinations showed all four dolphins had toxoplasmosis with tissue cysts resembling T gondii in the brain. Tachyzoite stages of T gondii were detected in the lungs, heart, liver, spleen and adrenal gland, variously of all four dolphins. Electron microscopy studies and immunohistochemistry confirmed the tissues cysts were those of Tgondii. All four dolphins also had intercurrent disease including pneumonia, three had peritonitis and one had pancreatitis. Conclusion: Four S chinensis necropsied in Queensland in 2000 and 2001 were found to be infected with toxoplasmosis. It is uncertain how these dolphins became infected and further studies are needed to determine how S chinensis acquire toxoplasmosis. All four dolphins stranded after periods of heavy rainfall, and coastal freshwater runoff may be a risk factor for T gondii infection in S chinensis. This disease should be of concern to wildlife managers since S chinensis is a rare species and its numbers appear to be declining.

A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

Spatial patterns of sub-tidal seagrasses and their tissue nutrients in the Torres Strait, northern Australia: Implications for management

The distribution and nutritional profiles of sub-tidal seagrasses from the Torres Strait were surveyed and mapped across an area of 31,000 km2. Benthic sediment composition, water depth, seagrass species type and nutrients were sampled at 168 points selected in a stratified representative pattern. Eleven species of seagrass were present at 56 (33.3%) of the sample points. Halophila spinulosa, Halophila ovalis, Cymodocea serrulata and Syringodium isoetifolium were the most common species and these were nutrient profiled. Sub-tidal seagrass distribution (and associated seagrass nutrient concentrations) was generally confined to northern-central and south-western regions of the survey area (

Clinical and pathological findings associated with congenital hypovitaminosis A in extensively grazed beef cattle

To determine the cause of exceptionally high mortality (41.4%) in perinatal calves on a beef cattle property 50 km south-west of Julia Creek in north-western Queensland. Investigations were based on clinical assessment of affected calves and laboratory analysis of pre- and postmortem specimens taken from 12 calves aged from 6 to 36 h of age. Associations between gross and histopathological findings and biochemical analyses conducted on serum and tissue samples were examined in relation to clinical observations. Clinical signs varied, but commonly included mild to severe ataxia, difficulty finding a teat and sucking, blindness (partial or complete, as judged by avoidance of obstacles) and depression with prominent drooping of the head. Gross and histopathological findings included herniation of the cerebellar vermis through the foramen magnum, squamous metaplasia of interlobular ducts in the parotid salivary glands and Wallerian degeneration of the optic nerves. Biochemical analysis of serum and liver samples available from four of the calves revealed low or undetectable levels of both vitamin A and vitamin E. Although vitamin E is known to have a sparing effect on vitamin A, the role (if any) played by deficiency of this vitamin was uncertain. The combination of clinical signs, postmortem findings, histopathological features and biochemical findings indicate that gestational vitamin A deficiency was highly likely to have been an important contributor to perinatal calf mortalities in this herd.

Raw data for project "Development of a DNA based aging technique for use in fisheries assessments" Fisheries and Development Corporation project 2007/033

The primary aim of this study was to determine the relationship between telomere length and age in a range of marine invertebrates including abalone (Haliotis spp) oysters (Saccostrea glomerata), spiny lobsters (Sagmariasus verreauxi formerly Jasus verreauxi and Jasus edwardsii) and school prawns (Metapenaeus macleayi). Additionally, this relationship was studied in a vertebrate organism using the freshwater fish Silver perch (Bidyanus bidyanus). Telomere length differences between tissues were also examined in some species such as Saccostrea glomerata, Sagmariasus verreauxi and Bidyanus bidyanus. In some cases cultured specimens of known age were used and this is quoted in the spreadsheets. For other wild-caught specimens where age was not known, size was used as a proxy for age. This may be a broad size class, or be determined by shell size or carapace length depending on the organism. Each spreadsheet contains raw data of telomere length estimates from Terminal Restriction Fragment Assays (TRF) for various individuals of each species including appropriate details such as age or size and tissue. Telomere length estimates are given in base pairs (bp). In most cases replicate experiments were conducted on groups of samples three times but on a small number of occasions only two replicate experiments were conducted. Further description of the samples can be found in final report of FRDC 2007/033. The arithmetic average for each individual (sample ID) across the two or three replicate experiments is also given. Bidyanus bidyanus (SilverPerch) Two sheets are contained within. a) Comparison of telomere length between different tissues (heart, liver and muscle) within the three year old age class - two replicate experiments were conducted. b) Comparison of telomere length between fish of different but known ages (0.25, 1, 2, and 3 years old) in each of three tissues, heart, liver and muscle – three replicate experiments were conducted per tissue. Haliotis spp (Abalone species) Three species were tested. H. asinina Telomere length was compared in two age classes-11 month and 18 month old abalone using muscle tissue from the foot. Within gel-variation was also estimated using a single sample run three times on one gel (replicate experiment). H. laevigata x H. rubra hybrids Telomere length was compared in three known age classes – two, three and four years old using muscle tissue from the foot. H. rubra Telomere length was compared in a range of different sized abalone using muscle tissue from the foot. Shell size is also given for each abalone Saccostrea glomerata Three sheets are contained within the file. a) Samples came from Moreton Bay Queensland in 2007. Telomere length was compared in two tissues (gill and mantle) of oysters in three age groups (1, 3 and 4 years) b) Samples came from Moreton Bay Queensland in 2009. Telomere length was compared in three age classes using DNA from gill tissue only c) Samples came from Wallis Lake, New South Wales. Telomere length was estimated from whole body minus the shell from 1 year old oysters, gill tissue of 3 age classes (1.5 years, 3 and 4 years), mantle tissue of two age classes (3 and 4 years). Sagmariasus verreauxi (formerly Jasus verreauxi) Telomere length was estimated from abdomen tissue of puerulus, gill and muscle tissue of 3 year old, large and very large size classes of lobsters. Jasus edwardsii Telomere length was measured in two size classes of lobsters- adults of varying sizes using muscle tissue and puerulus using tissues from the abdomen minus the exoskeleton. Metapenaeus macleayi Telomere length was measured in three size classes of school prawns adults. Muscle tissue was used, minus the exoskeleton.

Strategic banana tissue culture industry development and biosecurity act

Application and development of activities based on in vitro technologies delivering research, industry development and biosecurity activities to sustain and improve the Australian banana industry.

Telomere dynamics in the Sydney rock oyster (Saccostrea glomerata): an investigation into the effects of age, tissue type, location and time of sampling.

Telomere length has been purported as a biomarker for age and could offer a non-lethal method for determining the age of wild-caught individuals. Molluscs, including oysters and abalone, are the basis of important fisheries globally and have been problematic to accurately age. To determine whether telomere length could provide an alternative means of ageing molluscs, we evaluated the relationship between telomere length and age using the commercially important Sydney rock oyster (Saccostrea glomerata). Telomere lengths were estimated from tissues of known age individuals from different age classes, locations and at different sampling times. Telomere length tended to decrease with age only in young oysters less than 18 months old, but no decrease was observed in older oysters aged 2-4 years. Regional and temporal differences in telomere attrition rates were also observed. The relationship between telomere length and age was weak, however, with individuals of identical age varying significantly in their telomere length making it an imprecise age biomarker in oysters.

First Report of a Toxic Nodularia spumigena (Nostocales/Cyanobacteria) Bloom in Sub-Tropical Australia. II. Bioaccumulation of Nodularin in Isolated Populations of Mullet (Mugilidae)

Fish collected after a mass mortality at an artificial lake in south-east Queensland, Australia, were examined for the presence of nodularin as the lake had earlier been affected by a Nodularia bloom. Methanol extracts of muscle, liver, peritoneal and stomach contents were analysed by HPLC and tandem mass spectrometry; histological examination was conducted on livers from captured mullet. Livers of sea mullet (Mugil cephalus) involved in the fish kill contained high concentrations of nodularin (median 43.6 mg/kg, range 40.8-47.8 mg/kg dry weight; n = 3) and the toxin was also present in muscle tissue (median 44.0 mu g/kg, range 32.3-56.8 mu g/kg dry weight). Livers of fish occupying higher trophic levels accumulated much lower concentrations. Mullet captured from the lake 10 months later were also found to have high hepatic nodularin levels. DNA sequencing of mullet specimens revealed two species inhabiting the study lake: M. cephalus and an unidentified mugilid. The two mullet species appear to differ in their exposure and/or uptake of nodularin, with M. cephalus demonstrating higher tissue concentrations. The feeding ecology of mullet would appear to explain the unusual capacity of these fish to concentrate nodularin in their livers; these findings may have public health implications for mullet fisheries and aquaculture production where toxic cyanobacteria blooms affect source waters. This report incorporates a systematic review of the literature on nodularin measured in edible fish, shellfish and crustaceans.

Benzyl isothiocyanate: maximising production in papaya tissue extracts

Abstract: Although mainly grown for its sweet flavoured fruit, papaya (Carica papaya) has also been used for pharmacological purposes for many years. The reasons for use are varied with one of the best known being its anti-fungal action. Benzyl isothiocyanate (BITC) is the constituent most often implicated in this activity. Isothiocyanates are formed when the enzyme myrosinase catalyses the hydrolysis of the non-bioactive glucosinolates. This occurs when cellular contents come into contact through chewing, cutting or during extraction processes in the laboratory. While this is common in Brassica vegetables, the glucosinolate-myrosinase system is rare in fruit, papaya being a notable exception. It contains benzyl glucosinolate (BG), the glucosinolate precursor of BITC, in significant quantities. Parameters that determine the amount of BITC formed are duration of hydrolysis, presence/absence of nitrile-specifier proteins and BG content of different cultivars and tissues. We experimented with differing BITC extraction solvents, with the intention of developing a low cost, natural anti-fungal extract based on under-utilised papaya tissues. The findings suggest that papaya seeds, particularly from quarter-ripe fruit, have the potential to produce the highest levels of BITC necessary. Furthermore, they compare well with the nitrile-specifier protein-containing garden cress seeds (Lepidium sativum). To utilise the papaya seeds as a BITC source, an organic solvent such as ethanol is required to extract the largely water-insoluble BITC from the hydrolysed papaya seed mixture.